Cocaine receptor binding ligands

Abstract

Novel compounds useful as intermediates in the synthesis of compounds having high affinity for specific cocaine receptors in the brain have the formula ##STR1## wherein r is (CH₂)_n CH₂ Y, and wherein Y is H or F, X is a pharmaceutically acceptable anion, and

wherein Sn (r¹, r², r³) is ortho, meta or para and wherein r¹, r², r³ are independently alkyl of 1-6 carbon atoms and n is an integer of 0-5.

Description

This application is a continuation-in-part application Ser. No. of 07/972,472, filed Mar. 23, 1993, now U.S. Pat. No. 5,413,779; and also a continuation-in-part application Ser. No. of 08/164,576, filed Dec. 10, 1993, now U.S. Pat. No. 5,496,953; which is in turn a continuation-in-part Ser. No. of 07/792,648, filed Nov. 15, 1991, now abandoned; which is in turn a continuation-in-part of application Ser. No. 07/564,755, filed Aug. 9, 1990, now U.S. Pat. No. 5,128,118 and also a continuation-in-part of PCT/US91/05553, filed Aug. 9, 1991, the national phase of which is U.S. Pat. No. 07/972,472, now U.S. Pat. No. 5,413,779; all of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention is directed to a class of binding ligands for cocaine and other receptors in the brain. Specifically, a novel family of compounds shows high binding specificity and activity, and, in a radiolabeled form, can be used to bind to these receptors, for biochemical assays and imaging techniques.

DISCLOSURE OF PARENT APPLICATIONS:

In U.S. application Ser. No. 07/564,755, there is disclosure of a family of compounds exhibiting particularly high specificity and affinity for cocaine receptors and other neurotransmitter receptors in the brain of the formula:

Where the broken line represents an optional chemical bond and the substituents at 2 and 3 may be at any position;

The iodo substituent may be at o, m, p, or multisubstituted; ##STR2##

R₁ =CH₃, CH₂ CH=CH2, (CH₂)_n C₆ H₅ n=1-4;

R₂ =CH₃, CH₂ H₅,CH₃ (CH₂)₃, (CH₃)₂ CH, C₆ H₅, C₆ H₅ CH₂, C₆ H₅ (CH₂)₂ ;

X=pharmacologically acceptable anion

Sites of specific interest included cocaine receptors associated with dopamine transporter sites.

Subsequently, in PCT/US91/05553, filed Aug. 9, 1991 (U.S. National Phase application Ser. No. 07/972,472) from which priority is claimed, and which is incorporated herein by reference, the values for R₁ and R₂ were expanded, such that R₁ may be an alkyl of 1-7 carbon atoms, CH₂ CR₃ ═CR₄ R₅ wherein R₃ -R₅ are each, independently C₁-6 alkyl, or phenyl compounds of the formula C₆ H₅ (CH₂)_y, wherein y=1-6. The PCT filing also reveals the affinity of these compounds for cocaine receptors associated with serotonin transporters, and confirms, fort he first time, that the in vitro binding reported in the earlier-filed application, is confirmed in in vivo testing. Specific disclosure for a variety of applications, including using the receptors in both PET and SPECT scanning, wherein either the iodine substituent, or one of the carbon groups is radioactive (I-123, 125 or 131 and C11) thus providing methods for scanning for the presence of specific cocaine receptors appears. Such scanning processes may be used to determine physiological conditions, such as Parkinson's Disease, to examine in general the density and distribution of specific cocaine receptors in various parts of the brain and/or body, to determine the efficacy of neurological treatments aimed at halting or reversing the degeneration of specific nerves in the brain, and screening drugs, such as antidepressant drugs.

The affinity of these compounds, as reported in the applications incorporated, is surprisingly high, and compared with prior art compounds, such as [³ H]WIN 35,428, the novel compounds of these applications exhibit extremely low IC₅0 values for binding inhibition.

Other compounds exhibiting this type of binding activity and specificity would further advance this art, making it possible to obtain better scanning, with higher reliability, and lower levels.

SUMMARY OF THE INVENTION

Applicants' invention resides in the discovery of a new family of compounds of the formula ##STR3## Wherein Y═CH₂ R₃, CO₂ R₂, CONRR¹, or ##STR4##

R₁ =hydrogen, C₁-5 alkyl, or fluorinated alkyl,

R₂ =hydrogen, C₁-6 alkyl, C₃-8 cycloalkyl, C₁-4 alkoxy, C₁-6 alkynyl, halogen or amine,

R₃ =OH, hydrogen, C₁-6 alkyl, C₃-8 cycloalkyl, C₁-4 alkoxy, Cl, Br, I, CN, NH₂, NHC₁-6 alkyl, NC₁-6 alkyl, OCOC₁-6 alkyl, OCOC₁-3 alkylaryl,

A=S, O or N

X=H, C₁-6 alkyl, C₃-8 cycloalkyl, C₁-4 alkoxy, C₁-6 alkynyl, halogen, amino, acylamido,

and

Z=H, I, Br, Cl, F, CN, CF₃ NO₂, N₃, OR₁, CO₂ NH₂, CO₂ R₁, C₁-6 alkyl, NR₄ R₅, NHCOF₅, NHCO₂ R₆,

wherein R₄ -R₆ are each C₁-6 alkyl,

R and R¹ are independently H, C₁-6 alkyl, C₁-6 alkene, C₁-6 alkyne, phenyl, phenyl substituted with 1-3 of C₁-6 alkyl, alkene, alkyl or alkoxy, C₁-6 alkoxy, phenoxy, amine, amine substituted with 1-2 of C₁-6 alkyl, alkene, alkyne, alkoxy or phenyl or phenoxy or R and R¹ may combine to form heterocyclic structure including pyrrolidinyl, piperidinyl and morpholino moieties, unsubstituted or substituted with 1-2 C₁-6 alkyl, alkene, alkyne or alkoxy groups.

These compounds exhibit usually high affinity for binding to receptors for the dopamine transporter site, as well as the serotonin transporter site, again based on inhibition of [³ H]WIN 35,428 binding. It will be immediately apparent that certain of the compounds embraced within this novel class exhibit iodine substituents, and thus can be made easily radioactive, by substitution of a radioactive iodine, according to the synthesis scheme set forth in the prior applications incorporated herein by reference. In those circumstances where no radioactive iodine is provided, the compounds may be made radioactive by selective use of at least one carbon that is radioactive, e.g., [¹1 C].

These compounds are concurrently prepared by means of a tri-alkyl tin intermediate of the formula ##STR5##

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1 is a chart of IC₅0 values for alkyl and fluoroalkyl substituted N-derivatives of RTI-55, measured against RTI-55, the standard compound. FIGS. 1 and 3 reflect graphed in vivo studies of kinetics of the binding compounds of the invention, in mice. FIGS. 4 and 5 reflect, in graph form, in vivo studies of the striatal/cerebellar ratio for RTI-310 and RTI-313, in mice.

DETAILED DESCRIPTION OF THE INVENTION:

The compounds of this invention can be prepared according to the synthesis methods described in the parent applications. Additionally, specific synthesis routes and examples are set forth herein.

3β-[3'-Iodo-4'-aminophenyl]tropan-2β-carboxylic Acid Methyl Ester Dihydrochloride (1a)

To a solution of 3β-[4'-aminophenyl]tropan-2β-carboxylic acid methyl ester (300 mg, 1.094 mmol) in glacial AcOH (15 mL) was added dropwise ICI (195 mg, 1.201 mmol) at room temperature for 3 h under N₂. After removal of solvent, the residue was diluted with H₂ O, and then the pH was adjusted to basic with concentrated ammonia solution. The mixture was extracted with CHCl₃ which was washed with water and brine. After drying over MgSO₄, the solvent was evaporated to an oily product which was purified by flash chromatography (hexane-ether, 4:1). The collected fraction was converted to HCl salt with HCl/ether to yield 140 mg (29%) of 3β-[3'-iodo-4'-aminophenyl]tropan-2β-carboxylic acid methyl ester dihydrochloride (1a):mp 170°-173°C; [α]²5 -90.9° (c 0.055, MeOH), ¹ H NMR (250 MHZ, CDCl₃) δ61.65 (m, 3), 2.09 (m, 2), 2.2 (s, 3, NCH₃).2.45 (m, 1), 2.75 (m, 1, H-2), 2.8 (m, 1, H-3), 3.33 (m, 1, H-5), 3.45 (m, 4, H-1, OCH₃), 3.95 (m, 2, NH₂), 6.65 (d, 1, J=8.7, ArH, 7.05 (dd, 1, J=8.7, J=1.5, ArH), 7.42 (d, J=1.5, 1, ArH).

Anal. Calcd for C₁6 H₂1 IN₂ O₂.HCL.H₂ O: C, 39.12; H, 5.13; N, 5.70. Found: C, 39.12, H, 5.16; N, 5.63.

3β-[3'-Iodo-4'-azidophenyl]tropan-2β-carboxylic Acid Methyl Ester Dihydrochloride (1b) To a solution of 3β-[3'-iodo-4'-aminophenyl]tropan-2β-carboxylic acid methyl ester dihydrochloride (1a) (90 mg, 0.1902 mmol) in 1 mL, of AcOH (3M) was added an aqueous solution of NaNO₂ (17.3 mg, 0.2661 mmol, in 0.5 mL of H₂ O) at 0°C After 30 min at this temperature NaN₃ (19 mg, 0.2754 mmol) in 0.5 mL of H₂ O was added dropwise to the reaction mixture and stirred for 30 min at 0°C, then 30 min at room temperature. After removal of all solvent by evaporation, the residue was dissolved in CHCl₃ and washed with H₂ O. The organic layer was dried over MgSO₄ and concentrated to give an oil which was converted to HCl salt to yield 64 mg (72.7%) of 3β-[3'-iodo-4'-azidophenyl]tropan-2β-carboxylic acid methyl ester hydrochloride (1b) as a yellowish solid: mp 140°-143° C.; [α]²5 -97.4° (c 0.115), MeOh); ¹ H NMR (250 MHz, CDCl₃)δ1.51-1.73 (m, 3) 2.07-2.16 (m, 2), 2.19 (s, 3, NCH₃) 2.47 (m, 1), 2.80-2.93 (m, 2), 3.32 (m, 1, H-5), 3.51 (s, 3, OCH₃), 3.54 (m, 1, H-1), 7.01 (d, 1, J-7.7, ArH), 7.28 (dd, 1, J=7.77, J=1, ArH), 7.60 (d, 1, J=1, ArH).

Anal. Calcd for C₁6 H₁9 IN₄ O₂.HCl.H₂ O: C, 39.98; H, 4.61; N, 11.65. Found: C, 39.96.

Alternative synthesis for related compounds will be apparent to those of ordinary skill in the art. Additional schemes follow hereinbelow.

In the disclosure of the parent application Ser. No. 07/972,472, now U.S. Pat. No. 5,413,779 tri-alkyl tin intermediates of structure ##STR6## wherein A is, e.g., (CH₂)_n CH₃ or fluoroalkyl and X is e.g. CH₃, are disclosed as useful intermediates in the preparation of the active cocaine receptor binding ligands of this invention. This class of intermediates is exemplified in the parent application by the trimethyl tin intermediate, used in the example to make (¹23 I] dRTI-55 by iodination using a sodium acetate buffer and chloramine-T.

The identity of the alkyl substituent on the tin moiety substituted on the phenyl ring is variable, and can be adjusted by the individual skilled in the art to meet various needs, that are known per se. Thus, increasing the size and bulkiness of the alkyl moiety may make separation of the precursor easier, if the end-product is not being made in situ. Smaller alkyl groups, and straight as opposed to branched chain, alkyl groups, may improve solubility or similar parameters. Characteristics can be further optimized by providing different identities for 1,2 or all 3 of the alkyl moieties. While lower alkyl moieties of 1-6 carbon atoms, branched or straight chain, can generally be used, preferably alkyls of 1-4 carbon atoms are employed. Within this group, improved separation, such as through a conventional Sep-Pak™ kit can be achieved by providing a tri-t-butyl tin intermediate, while improved solubility is generally obtained through use of the trimethyl intermediate. Other alternatives, such as diethyl, t-butyl and methyl, ethyl, propyl tin intermediates can be used to achieve optimization of specific target parameters. As a general expression, compounds of the formula ##STR7## wherein R¹, R², and R³ are independently alkyl, straight or branched chain, of 1-6 carbon atoms, A is hydrogen, C₁-6 alkyl or fluoroalkyl, X is COOY or CONR⁴ R⁵, Y is (CH₂)_n CH₃ with n integer of 0-5, and R⁴, R⁵ are independently C₁-3 alkyl can be used as effective intermediates in the preparation of the binding ligands of this invention.

Synthesis

Treatment of 3β-(4-aminophenyl)tropan-2β-carboxylic acid methyl ester (1) with the appropriate halogen gives 2. Diazotization of 2 followed by the addition of sodium azide provides the 3-halo-4-azido analog 3 (Scheme 1).

Condensation of anhydroecgonine methyl ester (4) with the appropriate acylamide oxime gives the oxadiazole 5. Addition of the appropriate aryl lithium to 5 gives the cocaine analog 6. The addition of the appropriate aryl magnesium halide to 4 gives the analog 7 (Scheme 2).

Hydrolysis of 8 gives the acid 9. Reduction of 9 with diborane gives 10. Treatment of 9 with thionyl chloride, followed by the appropriate amine gives 11. Treatment of 10 with thionyl halide or acylating agent gives 12 and 13, respectively (Scheme 3). ##STR8##

Experimental

2-[3-Methyl-1, 2, 4-oxadiazol-5-yl]-8-methyl-8-azabicyclo[3.2.1]oct-2-ene (5, R═CH₃)

Acetamide oxime (500 mg, 6.75 mol) suspended in THF (50 mL) under nitrogen was heated at 60°C with NaH (132 mg, 5.5 mmol in oil dispersion) for 1 h. Anhydroecgonine methyl ester (2.76 ml) and 4 A° molecular sieves (2 g) were added and the reaction mixture heated under reflux for 3 h. After cooling, the reaction mixture was filtered and the solvent removed in vacuo. The residue was chromatographed on a silica gel column eluting with CHCl₃ --CH₃ OH (95:5) to give the free base.

3β-Phenyl-2β- (1,2,4-oxadiazonyl-5-methyl-3-yl)-tropane (6, R═CH₃, X═H)

To an oven-dried, round-bottomed flask equipped with rubber septum and nitrogen inlet was added dry THF (25 mL) and the oxadiazole (5, R═CH₃) (26 mg, 1.27 mmol), The reaction vessel was cooled to -78°C before adding phenyl lithium (0.636 mL, 1.27 mmol) of a 2M Et₂ O solution. The reaction mixture turned dark yellow. Stirring was continued for an additional 2 h before adding brine (10 mL). The crude mixture was extracted with chloroform (3×45 mL), and the combined organic layers were dried (MgSO₄) and concentrated under reduced pressure to yield a yellow solid. Recrystallization from hexanes gave 53 mg (39%) of pure product as white crystals: mp 124°-125°C; [α]_D +32.1° (c 0.14, MeOH); ¹ H NMR (250 MHz, CDCl₃) δ1.55-1.84 (m, 2), 1.88-1.98 (m, 2), 1.99-2.18 (m, 1), 2.24 (s, 3), 2.33 (s, 3), 2.47-2.58 (m, 1), 3.31-3.37 (t, 1, J=7.0 Hz), 3.56-3.62 (t, 1, J=5.7 Hz), 3.69-3.79 (ABq, 1, J=16.1, 8.0 Hz), 4.16-4.22 (t, 1, J=7.5 HZ), 7.0-7.14 (m, 5).

Anal. Calcd for C₁7 H₂1 N₃ O: C, H, N.

3β-(3-Bromo-4-aminophenyl)tropane Carboxylic Acid Methyl Ester (3, X═Br)

To a round-bottomed flask containing N,N-dimethylformamide (2.5 mL) was added 3β-(4-aminophenyl)tropane carboxylic acid methyl ester (100 mg, 0.365 mmol) and N-bromosuccinimide (64.5 mg, 0.365 mmol) under a stream of nitrogen gas at ambient temperature. The resulting solution immediately turned deep red. After stirring for an additional 2.5 h, water (5 mL) was added, and the crude reaction mixture was extracted with chloroform (3×25 mL). The combined organic extract was dried (MgSO₄) and concentrated under reduced pressure to yield the product as a brown oil. Flash chromatography (5% methanol-chloroform) afforded 42 mg (33%) of pure product as a yellow oil: mp of HCl salt 194°C dec; [α]_D -87.7° (c 0.09, MeOH); ¹ H NMR (250 MHz, DMSO) δ2.51-2.38 (m, 4), 3.39 (s, 3), 3.66-3.77 (td, 2, J=12.5, 2.9 Hz), 4.17-4.59 (br s, 2), 4.67 (s, 3), 4.69-4.96 (br s, 2), 5.92 (br s, 2), 7.96-8.68 (m, 3H).

Anal. Calcd for C₁6 H₂1 BrN₂ O₂.2 HCl.2 H₂ O): C, H, N.

General Procedure for Hydrolysis of 3β-[4-Halophenyl]tropan-2β-carboxylic Acid Methyl Esters

The methyl ester (1.0 mmol) was dissolved in 20 mL 50% aqueous dioxane and heated to reflux. After 6 h, the solvent was evaporated and the residue crystallized from MeOH--Et₂ O except as noted.

3β-[4-Iodophenyl]tropan-2β-carboxylic Acid (9, X═I)

The starting methyl ester (0.52 mmol, 0.20 g) gave 0.137 g (71%) of the acid as a white solid: mp, 318°-320°C; [α]_D²5 -79.3 (c 0.55, CH₃ OH); ¹ H NMR (CDCl₃) δ1.78 (m, 1), 2.02 (m, 2), 2.34 (m, 2), 2.61 (s, 3, --NCH₃), 2.7 (m, 3.12 (m, 1)3.73 (m, 2), 7.03 (d, 2, ArH), 7.62 (d, 2, ArH).

Anal. Calcd for C₁5 H₁8 INO₂ : C, 48.53; H, 4.89; N, 3.77. Found: C, 48.42; H, 4.89; N, 3.71.

3β-[4-Bromophenyl]tropan-2β-carboxylic Acid (9, X═Br)

The starting ester (0.38 g, 1.1 mmol) gave 0.208 g (58%) of the acid as a white solid: mp 304°-305°C; [α]_D -85.1° (c 0.55, CH₃ OH); ¹ H NMR (CDCl₃) δ1.79 (m, 1), 2.05 (m, 2), 2.33 (m, 2), 2.65 (s, 3, --NCH₃), 2.76 (m, 2), 3.315 (m, 1), 3.77 (m, 2), 7.16 (d, 2, ArH), 7.42 (d, 2, ArH).

Anal. Calcd for C₁5 H₁8 BrNO₂ : C, 55.57; H, 5.59; N, 4.32; Br, 24.65. Found: C, 55.36; H, 5.63; N, 4.28; Br, 24.53.

3β-[4-Fluorophenyl]tropan-2β-carboxylic Acid (9, X═F)

The starting ethyl ester (0.60 g, 2.2 mmol) gave 0.360 g (62%) of the acid as a white solid: mp 299°-300°C; [α]_D²5 -92.5° (ε0.89, CH₃ OH); ¹ H NMR (CDCl₃) δ1.80 (m, 1), 2.06 (m, 2), 2.36 (m, 2), 2.66 (s, 3, --NCH₃), 2.69 (m, 1), 2.79 (m, 1), 3.18 (m, 1), 3.79 (m, 2), 6.99 (m, 2, ArH), 7.25 (m, 2, ArH).

Anal. Calcd for C₁5 H₁8 FNO₂ : C, 68.42; H, 6.89; N, 5.32. Found: C, 68.29; H, 6.93; N, 5.26.

3β-[4-Chlorophenyl]tropan-2β-carboxylic Acid (9, X═Cl)

The starting ethyl ester (5.0 g, 6.91 mmol) gave 3.5 g (74%) of the acid (from H₂ O) as a white solid: mp 300°-301°C; [α]_D²5 -108.0° (c 0.10, CH₃ OH); ¹ H NMR (CDCl₃) δ1.57-1.9 (m, 4), 2.25 (m, 2), 2.45 (s, 3, NCH₃), 2.52 (m, 1), 3.12 (m, 1, H-2), 3.55 (m, 2, H-1, H-5), 7.19 (dd, 4, ArH).

Anal. Calcd for C₁5 H₁8 ClNO₂∅25 H₂ O: C, 63.38; H, 6.56; N, 4.93. Found: C, 63.78; H, 6.56; N, 4.97.

General Procedure for Preparation of 3β-[4-Halophenyl]-2β-hydroxy-methyltropane

The 2β-carboxylic acid (1.0 mmol) was suspended in dry THF (20 mL) at 0°C under N₂. A solution of BH₃ in THF (4.0 mL of 1M solution, 4.0 mmol) was added by syringe. After 3 h, the reaction was quenched with conc. HCl (1.0 mL) and stirred for 30 min. The solvent was evaporated and the residue partitioned between dilute NH₄ OH and CH₂ Cl₂. The aqueous phase was further extracted with CH₂ Cl₂ (3×50 mL). The organic extract was dried over Na₂ SO₄, filtered and evaporated leaving a white solid. This was chromatographed on a silica gel flash column eluting with Et₂ O--Et₃ N (9:1). The sample from the column was crystallized from pentane, except as noted.

3β-[4-Iodophenyl]-2β-hydroxymethyltropane (10, X═I)

The starting 2β-carboxylic acid (0.100 g, 0.270 mmol) gave 0.055 g (57%) of the product as a white crystalline solid: mp 104°-105°C: [α]_D²5 -54.6 (c 0.5, CHCl₃): ¹ H NMR (CDCl₃)δ1.46 (m, 1), 1.66 (m, 1), 1.7 (d, 2), 2.17 (m, 2), 2.27 (s, 3, NCH₃), 2.48 (m, 1), 3.03 (m, 1), 3.34 (m, 2), 3.45 (m, 1), 3.75 (m, 1), 7.13 (d, 2, ArH), 7.63 (d, 2, ArH).

Anal. Calcd for C₁5 H₂0 INO: C, 50.43; H, 5.64; N, 3.92. Found: C, 50.52: H, 5.67; N, 3.84.

3β-[4-Bromophenyl]-2β-hydroxymethyltropane (10, X═Br)

The starting 2β-carboxylic acid (0.150 g, 0.463 mmol) gave 0.045 g (315) of the product as a white crystalline solid: mp 92°-93°C: [α]_D²5 -55.8° (c 0.5, CHCl₃); ¹ H NMR (CDCl₃) δ1.46 (m, 1), 1.62 (m, 1), 1.72 (d, 2), 2.17 (m, 2), 2.27 (s, 3, NCH₃), 2.50 (m, 1), 3.03 (m, 1), 3.34 (m, 2), 3.45 (m, (1), 3.76 (m, 1), 7.25 (d, 2, ArH), 7.43 (d, 2, ArH).

Anal. Calcd for C₁5 H₂0 BrNO: C, 58.07; H, 6.50; N, 4.52; Br, 25.76. Found: C, 57.97: H, 6.55: N, 4.45: Br, 25.83.

3β-[4-Fluorophenyl]-2β-hydroxymethyltropane (10, X═F)

The starting 2β-carboxylic acid (0.263 g, 1.0 mmol) gave 0.140 g (56%) of the product as a white crystalline solid: mp 79°-80°C: [α]_D²5 -59.8° (c 0.5, CHCl₃): ¹ H NMR (CDCl₃) δ1.45 (m, 1), 1.63 (m, 1), 1.72 (d, 2), 2.16 (m, 2), 2.27 (s, 3, NCH₃), 2.49 (m, 1), 3.07 (m, 1), 3.34 (m, 2), 3.45 (m, 1), 3.76 (m, 1), 6.99 (m, 2, ArH), 7.32 (m, 2, ArH).

Anal. Calcd for C₁5 H₂0 FNO: C, 72.26; H, 8.08; N, 5.62. Found: C, 72.17; H, 8.10: N, 5.61.

3β-[4-Chlorophenyl]-2β-hydroxymethyltropane (10, X═Cl)

The starting 2β-carboxylic acid (0.950 g, 3.4 mmol) gave 0.30 g (33%) of the product as an off-white crystalline solid: mp 104°-106°C: [α]_D²5 -82.4° (c 0.21, CH₃ OH); ¹ H NMR (CDCl₃) δ1.45 (m, 1), 1.67 (m, 3), 2.17 (m, 2), 2.25 (s, 3, NCH₃), 2.50 (m, 1), 3.05 (m, 1, H-3), 3.30 (m, 2), 3.40 (m, 1, H-1), 3.72 (dd, 1), 7.29 (m, 4, ArH).

Anal. Calcd for C₁5 H₂0 ClNO: C, 67.78; H, 7.59; N, 5.27. Found: C, 67.63; H, 7.69; N, 5.25.

3β-[p-Chlorophenyl]-2β-acetoxymethyltropane (13, X═Cl)

To a flask containing acetic anhydride (10 mL) in dry pyridine (5 mL) at ambient temperature was added 3β-(p-chlorophenyl)-2β-hydroxymethyltropane (95 mg, 0.32 mmol). The reaction mixture was maintained at room temperature for 2 h before diluting with water (10 mL) and adjusting the pH of the aqueous phase to 14. After extraction of the aqueous phase with chloroform (3×25 mL), the organic layers were combined, dried (MgSO₄) and concentrated under reduced pressure to yield the crude product as a yellow oil. Flash chromatography (CHCl₃ --MeOH, 9:1) yielded 45 mg (41%) of pure product as a colorless oil: mp of HCl salt 202°C dec; [α]_D -57.1° (c 0.070, MeOH); ¹ H NMR (250 NHz, CDCl₃) δ2.02 (s, 3), 2.17-2.59 (m, 6), 2.87 (s, 3), 3.49-3.69 (m, 2), 3.99-4.22 (m, 4), 7.27-7.41 (m, 4).

Anal. Calcd for C₁7 H₂2 ClNO₂.HCl∅25 H₂ O: C, H, N.

3β-(p-Chlorophenyl)-2β-(N-methylcarbamoyl)tropane (11, R═CH₃, R₂ ═H, X═Cl)

To a flask containing thionyl chloride (10 mL) at 0°C was added 3β-(p-chlorophenyl)tropane-2β-carboxylic acid (183 mg, 0.0715 mmol). The mixture was maintained at 0°C for 4 h before concentrating under reduced pressure. The brown residue was dissolved in ethylene chloride (10 mL) and cooled to 0°C before adding methylamine (5 mL). Stirring was continued for 15 min after which the excess methylamine was allowed to evaporate. The brown residue was diluted with water (25 mL) and extracted with CHCl₃ (3×25 mL). The combined extracts were dried (MgSO₄) and concentrated under reduced pressure to give the crude product as a brown oil. Flash chromatography (CHCl₃ --MeOH, 9:1) yielded 72 mg (37%) of pure product as a yellow oil: mp HCl salt 138°C; [α]_D -96.9° (c 0.170, MeOH); ¹ H NMR (250 MHz, CDCl₃) δ1.55-1.88 (m, 5), 2.07-2.28 (m, 2), 2.31 (s, 3), 2.35-2.55 (m, 1), 2.69 (s, 3), 3.11-3.33 (m, 1), 3.40-3.49 (br s, 1), 7.14-7.26 (m, 4).

Anal. Calcd for C₁6 H₂1 ClN₂ O.HCl∅75 H₂ O): C, H, N.

3β-(p-Chlorophenyl)-2β-chloromethyltropane (12, X═Y═Cl)

To a flask containing thionyl chloride (5 mL) was added 3β-(p-chlorophenyl)-2β-hydroxymethyltropane (64 mg, 0.24 mmol). The reaction mixture was maintained at reflux for 2 h before carefully diluting with water and adjusting the pH of the aqueous phase to 14 with conc. ammonium hydroxide. The aqueous layer was extracted with CHCl₃ (3×25 mL). The organic layers were combined, dried (MgSO₄), and concentrated under reduced pressure to yield the crude product as a brown oil. Flash chromatography (CHCl₃ --NeOH, 9:1) yielded 33 mg (52%) of pure product as a colorless oil: mp of HCl salt 208°C; [α]_D =63.9° (c 0.155, MeOH); ¹ H NMR (250 MHz, CDCl₃) δ1.05-2.50 (m, 6), 2.69 (s, 3), 2.88-3.16 (m, 2), 3.25-3.52 (m, 1), 3.78-3.89 (br s, 1), 4.02-4.15 (br s, 1), 4.55 (t, 1, J=12.3 Hz), 7.01-7.59 (m, 4).

Anal. Calcd for C₁5 H₁9 Cl₂ N.HCl: C, H, N.

3β-(3,4-Dichlorophenyl)-2β-chloromethyltropane (7, X═Y═Cl)

To a three-neck, round-bottomed flask containing freshly distilled ether (125 mL) and magnesium turnings (268 mg, 11.0 mmol),was added 3,4-dichloroiodobenzene (2.26 g, 8.27 mmol). After 2 h, the reaction flask was equipped with a mechanical stirrer, and the Grignard reagent was cooled to -55°C before adding anhydroecgonine methyl ester (500 mg, 2.75 mmol). The resulting solution was stirred for an additional 2.5 h before being cooled to -78°C After 1 h, 2 mL of trifluoroacetic acid was added to the solution followed by 2 h of stirring. The quenched reaction mixture was then diluted with 1N HCl (100 mL) and extracted with ether (3×100 mL). The ethereal layers were discarded, and the aqueous layer was basified with conc. ammonium hydride and then extracted with chloroform (3×50 mL). The combined organic layers were dried (MgSO₄) and concentrated under reduced pressure to yield the crude product as a colorless oil. Flash chromatography (ether triethylamine, 9:1) yielded 71 mg (9.0%) of pure product: 1H NMR (250 MHz, CDCl₃) δ1.52-1.76 (m, 2), 1.81-1.95 (m, 2), 1.96-2.22 (m, 2), 2.38 (s, 3), 3.07-3.15 (br s, 2), 3.21-3.32 (br s, 1), 3.45-3.65 (m, 1), 3.50 (s, 3), 7.10-7.38 (m, 3).

Anal. Calcd for C₁6 H₁9 Cl₂ NO₂.HCl): C, H, N.

3β-(4-Chloro-3-methylphenyl) -2β-chloromethyltropane (7, X═Cl, Y═CH₃)

To a three-neck, round-bottomed flask containing freshly distilled ether (125 mL) and magnesium turnings (200 mg, 8.25 mmol) was added 4-chloro-3-methylbromobenzene (1.69 g, 8.25 mmol). After 2 h, the reaction flask was equipped with a mechanical stirrer, and the Grignard reagent was cooled to -55°C before adding anhydroecgonine methyl ester (500 mg, 2.75 mmol). The resulting solution was stirred for an additional 2.5 h before being cooled to -78°C After 1 h, 2 mL of trifluoroacetic acid was added to the solution followed by 2 h of stirring. The quenched reaction mixture was then diluted with of 1N HCl (100 mL) and washed with ether (3×100 mL). The aqueous layer was basified with conc. ammonium hydroxide and extracted with CHCl₃ (3×50 mL). The combined organic layers were dried (MgSO₄) and concentrated under reduced pressure to yield the crude product as a colorless oil. Flash chromatography (ether-triethylamine, 9:1) yielded 45 mg (5.0%) of pure product: ¹ H NMR (250 MHz, CDCl₃) δ1.51-1.83 (m, 2), 1.97-2.21 (m, 2), 2.20 (s, 3), 2.45-2.59 (td, 1, J=9.5, 2.6 HZ, 2.82-3.02(m, 3), 3.34-3.40 (br s, 1), 3.51 (s, 3), 3.52-3.61 (br s, 1), 7.00-7.23 (m, 3).

Anal. Calcd for C₁7 H₂2 ClNO₂.HCl.2 H₂ O: C, H, N.

3β-(3'-Methyl-4'-fluorophenyl)tropan-2β-carboxyltc Acid Methyl Ester (7, X═F, Y═CH₃)

The title compound was prepared by modification of a reported procedure used to prepare other similar compounds.^ref Thus, using anhydroecgonine methyl ester (500 mg, 2.76 mmol) and 3-methyl-4-fluorophenyl magnesium bromide (prepared from 200 mg of magnesium metal and 1 mL of 3-methyl-4-fluoro-1-bromobenzene) yielded 234 mg (29%) of the title compound. The hydrochloride salt had mp 163°-165°C; [α]_D²5 -103.8° (c 0.08, MeOH); ¹ H NMR of free base of 41 (250 MHz, CDCl₃) δ1.67 (m, 3), 2.15 (m, 2), 2.19 (s, 3, CH₃), 2.20 (s, 3, NCH₃), 2.55 (m, 2), 2.87 (m, 1, H-2), 2.93 (m, 1, H-3), 3.35 (m, 1, H-5), 3.49 (s, 3, OCH₃), 3.55 (m, 1, H-1), 6.85, 6.97 (m, 3, C₆ H₃).

Anal. Calcd for C₁7 H₂3 CIFNO₂.1.5 H₂ O: C, 57.54; H, 7.39; N, 3.95. Found: C, 57.88: H, 7.21; N, 4.20.

[³ H]WIN 35,428 Radioligand Binding

Rat striata from male Sprague-Dawley rats (250-350 g) were rapidly dissected, frozen, and stored at -70°C until used. The frozen rat striata were homogenized in 20 volumes of 10 mM phosphate buffer (pH 7.4) containing 0.32M sucrose using a polytron (setting 6) for 10 sec. The homogenate was centrifuged for 10 min at 50,000 x g, the resulting pellet was washed in buffer, recentrifuged, and resuspended to a tissue concentration of 10.0 mg/mL. Binding assays were carried out in a total volume of 0.5 mL containing 0.5 nM [³ H]WIN 35,428 and 1.0 mg tissue. The suspensions were incubated for 2 h on ice. Incubations were terminated by filtration with three 5 mL washes through Whatman GF/B filters previously soaked in 0.05% polyethylenimine using a Brandel N48R filtering manifold (Brandel Instruments, Gaithersburg, Md.). Radioactivity was counted in 5 mL of scintillation cocktail in a Beckman LS 3801 liquid scintillation counter with an efficiency of approximately 50%. Nonspecific binding of [³ H]WIN 35,428 was defined by the presence of 30 μM (-)-cocaine. Under these conditions, nonspecific binding was approximately 5-8% of total binding. IC₅0 values were determined from competition curves of 10-12 points utilizing the curve fitting program EBDA. Mean values and standard errors were calculated from 3-4 assays for each test drug.

Tissue Preincubation with Irreversible Agents

Tissue was prepared as described above, and the final homogenate was incubated for 60 min with either drug or vehicle as control for 60 min on ice in the above buffer. Following the 60 min incubation period, all compounds containing an azido group were then exposed to UV light (2800 Å) for 40. sec. The incubation of all compounds was terminated by centrifugation at 50,000 x g for 10 min. The resulting pellet was resuspended to a concentration of 10 mg/mL, and an aliquot was removed (0 washes). This procedure was repeated for a total of 3 washes. Residual [³ ]WIN 35,428 binding was determined as described above. Data are expressed as the percent of specific control binding.

Testing of various compounds within the described class has given remarkably high binding values. Thus, as reported in the parent applications, receptor binding activity can be determined by degree of inhibition of the binding of [³ H]WIN 35,428. In such assays, the ligand of interest is assigned a IC₅0 value, when incubated in a 10 nM phosphate buffer, pH 7.4, containing 0.32^m sucrose, with 0.5 nM [³ H]WIN 35,428 for a two hour period of time. After that, the radioactivity bound to the substrate is measured. As reported in U.S. application Ser. No. 07/564,755, now U.S. Pat. No. 5,128,118 on binding to a dopamine transporter receptor site, cocaine gave a IC₅0 of 89.00 nM, WIN 35,428 gave a value of 14.00 nM and a compound representative of the subject matter claimed in that application, 3β-[4-iodophenyl]-tropane-2β-carboxylic acid methyl ester tartrate gave a IC₅0 value of 0.25nM. In similar assays, values of 1.35 and 4.93 nM were obtained for compounds within the class set forth above, particularly, those compounds bearing a carboxylic acid moiety, or a heterocyclic moiety. Compounds having the structure of compound 11 of synthetic scheme 3 have been prepared and tested as reflected in Table II. Similar values may be obtained for the remaining members of the class.

When bearing an appropriate radioactive label, such as ¹1 C or ¹25 I, ¹25 I or ¹31 I, these compounds, preferential binders to dopamine transporter and serotonin transporter binding sites, can be used as imaging agents for both positron emission tomography (PET) as well as single photon emission computed tomography (SPECT). PET may require the [¹1 C] labeled form of the drug, while radioactive iodine-labeled compounds may be used in SPECT scanning.

As noted, such scanning has a variety of utilities. The actual density and distribution of cocaine receptors in various parts of the brain and CNS is of interest, and can be mapped through the use of these compounds. Additionally, as noted above, identification of degeneration of nerve terminals, corresponding to a loss of dopamine transporter sites, can be determined by this scanning, to diagnose Parkinson's Disease. In addition, progression of the disease, and efficacy of treatment, can be monitored by such scanning. Similarly, degeneration of specific nerves in the brain due to exposure to various toxins can be detected or monitored by the scanning made possible by these compounds.

As an additional use, drugs having high affinity for the transporter sites bound to by these compounds, particularly serotonin and dopamine transporter sites, can be screened, using these compounds, in the identified scanning methods. The scanning itself is conventional, given the use of these compounds, and does not constitute an aspect of the invention per se. Affinity values for representative compounds are given in the following table.

TABLE I
______________________________________
Potencies of Cocaine and Analogs in Inhibiting Binding of
[3 H]-3β-(4-Fluorophenyl)tropan-2β-carboxylic Acid Methyl
Ester (WIN 35,428)
Compound            IC50 (nM)
______________________________________
Cocaine         102
2       (X = I)         1.35
3       (X = I)         4.93
6       (R = CH3, X = H)
48
7       (X = Y = Cl)    0.79
7       (X = Cl, Y = CH3)
0.81
9       (X = Br)        279
9       (X = I)         474
9       (X = Cl)        2070
9       (X = F)         2740
10      (X = Br)        1.49
10      (X = Cl)        1.53
10      (X = I)         2.2
10      (X = F)         47.3
11      (R1 = CH3, R = H, X = Cl)
12.4
12      (X = Y = Cl)    2.64
13      (R = CH3, X = Cl)
1.6
______________________________________

As noted, the substituent on the nitrogen group may be hydrogen, alkyl or fluoroalkyl. Alkyl and fluoroalkyl-substituted compounds give closely related properties. Propyl and F-propyl N-derivatives in particular have been tested to give equivalent values. Thus, the F-propyl N-derivative and propyl N-derivative of RTI-55 give IC₅0 values of 1.17 and 1.67 and M respectively. The data is reported in FIG. 1.

In vivo binding testing also demonstrates similarities between the class of compounds identified. As set forth in FIGS. 2 and 3, subsequent to IV injection, both compounds get into the brain rapidly and show regional differences at the earliest time measured--20 minutes. The radioactivity distributed according to the distribution of dopamine transporters, as expected. Both compounds had very similar properties. RTI-310 left the striatal compartment a little bit more slowly than RTI-313, possibly reflecting a slightly higher affinity for the transporter.

FIGS. 4 and 5 show the striatal to cerebellar ratio for RTI-310 and 313, respectively. Again, the results are quite similar. Thus, while the ratio for RTI-313 may be maintained slightly longer than that for RTI-310, RTI-310 achieves a slightly higher ratio than RTI-313. Both the alkyl and fluoroalkyl-substituted compounds are acting in the same manner, and prove to have similar utility as imaging agents particularly between 45 and 180 minutes after injection. In vitro data were measured from rat tissue studies, while the in vivo data are derived from mice.

This invention has been described in both generic terms, and by reference to specific description. No specific description or example is considered binding, unless so identified. Alternate forms and methods will occur to those of ordinary skill in the art, without the exercise of inventive faculty, and remain within the scope of this invention, save as limited by the claims set forth below.

TABLE II
__________________________________________________________________________
Binding Data for 3β-(Substituted Phenyl)tropan-2β-carboxylic
Amides
##STR9##
Code                    DA    NE(N)  5-HT    NE/DA
5-HT/DA
Name R           R'  X  IC50  (nM)
IC50  (nM)
IC50  (nM)
Ratio
Ratio
__________________________________________________________________________
RTI-106
CH3    H   Cl 12.4 ± 1.18
1511 ± 23
1312 ± 46
122 106
RTI-118
H           H   Cl 11.5 ± 1.62
4267 ± 359
1621 ± 110
371 140
RTI-129
CH3    CH3
Cl 1.38 ± 0.1
942 ± 48
1079 ± 102
682 782
RTI-146
CH2 OH H   Cl 2.05 ± 0.23
144 ± 3
97.8 ± 10.3
70  48
RTI-147
CH2 CH2 CH2 CH2
Cl 1.38 ± 0.03
3949 ± 72
12,394 ± 1207
2861
8981
RTI-156
CH2 CH2 CH2 CH2 CH2
Cl 6.61 ± 1.15
5832 ± 791
3468 ± 266
882 524
RTI-170
CH2 CCH
H   Cl 16.5 ± 1.32
1839 ± 112
4827 ± 158
112 292
RTI-172
NH2    H   Cl 44.1 ± 4.6
3914 ± 127
3815 ± 238
89  87
RTI-174
NHCOCH3
H   Cl 157.7 ± 11
43,515 ± 596
125,177 ± 8280
276 793
RTI-182
CH2 COC6 H5
H   Cl 7.79 ± 0.62
1722 ± 148
827 ± 48
221 106
RTI-183
OCH3   CH3
Cl 0.85 ± 0.06
549 ± 19
724 ± 94
645 851
RTI-198
CH2 CH2 CH2
Cl 6.57 ± 0.67
990 ± 4.8
813 ± 57
150 123
RTI-196
CH3 O  H   Cl 10.7 ± 1.2
9907 ± 631
43,677 ± 1960
925 4082
RTI-201
NHCOC6 H5
H   Cl 91.83 ± 15.4
20,731 ± 935
48,810 ± 4775
225 531
RTI-208
OCH2 CH2 CH2
Cl 1.47 ± 0.13
998 ± 26
2470 ± 56
678 1680
RTI-214
CH2 CHOCH2 CH2
Cl 2.90 ± 0.3
88,768 ± 1854
30,609
RTI-215
C2 H5
C2 H5
Cl 5.48 ± 0.19
9432 ± 770
1721
RTI-217
3'-OHC6 H4
H   Cl 4.78 ± 0.44
30,976 ± 334
16,827 ± 1540
6480
3520
RTI-218
OCH3   CH3
Cl 1.19 ± 0.09
520    1911 ± 103.5
437 1605
RTI-226
C6 H5
CH3
Cl 45.04 ± 3.05
23,926 ± 3527
525
RTI-133
H           H   CH3
41.8 ± 2.45
4398 ± 271
6371 ± 374
105 152
RTI-166
NHCOCH3
H   CH3
543 ± 79
>10,000
>10,000 >18 >18
RTI-168
CH2 CHCH
H   CH3
56.2 ± 6.9
11,087 ± 553
14,878 ± 959
197 265
RTI-169
NH2    H   CH3
84.5 ± 6.8
5970 ± 474
37,604 ± 3128
71  445
RTI-175
CH2 COC6 H5
H   CH3
22.8 ± 0.88
2117 ± 116
4395 ± 87.8
93  193
RTI-186
CH3    OCH3
CH3
2.55 ± 0.43
442 ± 26
3402 ± 353
173 1334
RTI-197
NHCOC6 H5
H   CH3
141.8 ± 9.77
37,852 ± 4144
>200,000
267 >1410
RTI-221
C2 H5
C2 H5
CH3
27.4 ± 1.93
8890   33,928 ± 2192
325 1238
RTI-227
OCH2 CH2 CH2
I  0.75 ± 0.02
446 ±
130 ± 15.8
594 173
RTI-228
OCH3   CH3
I  1.08 ± 0.15
92.5 ± 17.55
86
RTI-229
CH2 CH2 CH2 CH2
I  0.37 ± 0.04
991 ± 20.9
1,728 ± 39.3
2678
4670
__________________________________________________________________________

Claims

1. A compound of the formula ##STR10## wherein r is (CH₂)_n CH₂ Y, and wherein Y is H or F, X is a pharmaceutically acceptable anion,

wherein SnR¹ r² r³, is ortho, meta or para and wherein r¹, r², r³ are independently alkyl of 1-6 carbon atoms and n is an integer of 0-5, and is a single or double bond.

2. The compound of claim 1, wherein r¹, r², and r³ are independently alkyl of 1-4 carbon atoms.

3. The compound of claim 2, wherein r¹, r², and r^{' are each t-butyl.}

4. The compound of claim 2, wherein r², r², and r³ are methyl, ethyl and propyl, respectively.

5. The compound of claim 1, wherein the compound has the formula ##STR11## wherein is a single bond or double bond.

6. The compound of claim 1, wherein r is --CH₂ CH₂ CH₃.

7. The compound of claim 6, wherein r¹, r² and r³ are independently alkyl of 1-4 carbon atoms.

8. The compound of claim 6, wherein each of r¹, r² and r³ are t-butyl.